Islet cell cluster produced from human umbilical cord mesenchymal stem cells

• US 9,017,657 B2
• Filed: 01/15/2009
• Issued: 04/28/2015
• Est. Priority Date: 07/16/2008
• Status: Active Grant

• Current Assignee: National Taiwan University (Government of The Republic of China)
• Original Assignee: National Taiwan University (Government of The Republic of China)
• Inventors: Liu, Shing-Hwa, Chao, Kuo-Ching, Chao, Kuo-Fang
• Primary Examiner(s): Crouch, Deborah
• Assistant Examiner(s): SGAGIAS, MAGDALENE K
• Application Number:
  US12/320,026
• Publication Number:
  US 20100015103A1
• Time in Patent Office: 2,294 Days
• US Class Current: 424/93.1
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin

Abstract

The invention relates to a cell culture method, particularly to a co-culture method for human mesenchymal stem cells and target animal cells, in order to solve the problem that animal cells are not easy to survive alone upon culturing. The invention also provides a method for using a stem cell conditioned medium to culture animal cells. The invention also provides a method to induce the transformation of human fetal islet-like cell clusters from human stem cells and its application thereof.

Claims

1. A method to induce transformation of human islet cell cluster from human mesenchymal stem cells, comprising,

(i) isolating human umbilical cord mesenchymal stem cells (HUMSCs) from human umbilical cord;

(ii) culturing the isolated HUMSCs in a medium containing serum;

(iii) culturing the cultured HUMSCs in mammalian neuronal cell conditioned medium (NCM) containing cytosine arabinoside (AraC) to produce nestin-positive cells;

(iv) culturing the nestin-positive cells in DMEM/F12 medium containing 2% serum, nicotinamide (NAD) and vitamin B27 to produce islet-like cell clusters (ICCs); and

(v) culturing the ICCs in DMEM/F12 medium containing 2% serum, NAD and vitamin B27 supplement with mesenchymal stem cell conditioned medium (SCM) containing one or more active components selected from the group consisting of interleukin-6 (IL-6), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), monocyte chemoattractant protein-1 (MCP-1), growth related oncogene (GRO), hepatocyte growth factor (HGF), insulin-like growth factor binding protein 4 (IGFBP-4) and interleukin-8 (IL-8) to produce ICCs expressing insulin and glucagon.

2. A cell culture method, comprising:

(i) preparing a mammalian NCM;

comprising

(a) culturing mammalian brain cells in a single cell suspension in a medium comprising AraC, wherein AraC inhibits glial cells to produce mammalian NCM; and

(b) harvest the medium;

(ii) culture HUMSCs in mammalian NCM to produce nestin-positive cells;

(iii) culture the nestin-positive cells in DMEM/F12 medium, glucose, insulin, NAD, and B27 to induce the nestin-positive cells to differentiate into premature ICCs;

(iv) culture the premature ICCs in in DMEM/F12 medium, glucose, insulin, NAD, B27 and SCM, wherein SCM contains one or more active components selected from the group consisting of interleukin-6 (IL-6), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue inhibitor of metalloproteinase-2 (TIMP-2), monocyte chemoattractant protein-1 (MCP-1), growth related oncogene (GRO), hepatocyte growth factor (HGF), insulin-like growth factor binding protein 4 (IGFBP-4) and interleukin-8 (IL-8) to induce transformation of premature ICCs into mature insulin secreting ICCS.

6. The method according to claim 2, wherein the concentration of glucose is 20 mM to 30 mM.

7. The method according to claim 2, wherein the concentration of glucose is 25 mM.

3. A method of inducing transformation of human islet cluster from human mesenchymal stem cells comprising;

(i) isolating HUMSCs;

(ii) culturing the isolated HUMSCs in a medium containing serum;

(iii) culturing the cultured HUMSCs from step (ii) in mammalian NCM containing AraC to produce nestin-positive cells; and

(iv) culturing the nestin-positive cells in DMEM/F12 medium containing 2% serum, NAD and vitamin B27 to produce ICCs.

4. The method according to claim 3, wherein the neuronal conditioned medium is the cell medium for culturing mammalian brain cells.

5. The method according to claim 3, wherein the nestin positive cells are cultured in DMEM/F12 medium comprising glucose, insulin, NAD, and B27.